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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 66 bp
Wild Type = 92 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
35115 | GAA GGG CTC ACT TTC TCA CG | Mutant Reverse | A | |||
45701 | GGG GTG GGG AAG AAA TAA TG | Common | A | |||
45702 | CCC CAG CTA TAT CCC TTT TTG T | Wild type Reverse | A | |||
45703 | Fluorophore-1 | TAA ACA GAA ACC ATA CTG ATT AGT TCT GA | Quencher-1 | WT Probe | ||
45704 | Fluorophore-2 | TAA ACA GAA ACT GTC AGA GAC CAT CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
35115 | 0.40 uM |
45701 | 0.40 uM |
45702 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |